Background. Human genetic polymorphisms in metabolic activation and detoxification pathways are a major source of inter-individual variation in susceptibility to environmentally induced disease. The group has developed genotyping assays for the "at-risk" variants of enzymes that protect against carcinogens in cigarette smoke, diet, industrial processes and environmental pollution. Population studies indicate that for these candidate susceptibility genes, the frequency of the at-risk genotypes for glutathione transferase M1 (GSTM1), theta 1 (GSTT1), Pi (GSTP1) and N-acetyltransferase (NAT1 and NAT2), XRCC1, XPD, vary significantly between ethnic groups. Some differences in cancer incidence among groups may be due to genetic metabolic differences as well as exposure differences. Mission: Our long-term goal is to understanding how genes and environment interact to influence risk of environmentally induced disease. To this end we are engaged in "Environmental Genomics." This encompasses: 1) identification of candidate environmental response genes, 2) discovery and functional characterization of genetic and phenotypic variation in these genes, and;3) the analysis in population studies of environmental disease susceptibility associated with functional polymorphisms, acquired susceptibility factors and exposures;and the interactions between these factors. Eventually we hope these genomic approaches will help us to develop assays using genotype, gene expression, and other biomarkers of exposure and effect, that will be predictive of future risk. This work has been extended to look at methylation of CpG sites in the human genome. This information will allow us to more carefully determine the bounds of human variability in risk assessment and will be useful in developing prevention strategies to reduce disease incidence. The Genetic Susceptibility Project takes the candidate susceptibility factors from the laboratory genotype/phenotype studies and tests them in population studies. We are collaborating with numerous NIH, and university-based epidemiology groups to design and carryout appropriate tests of these factors in population-based epidemiology studies. Progress/accomplishments: 1) Genetically determined factors that alter the metabolism of tobacco carcinogens can influence an individuals susceptibility to bladder cancer. The associations between the genotypes of glutathione S-transferase (GST) M1, GSTP1, GSTT1 and N-acetyl-transferase (NAT) 1, and the phenotypes of NAT2 and cytochrome P450 (CYP) 1A2 and bladder cancer risk were examined in a case-control study involving 731 bladder cancer patients and 740 control subjects in Los Angeles County, California. Individual null/low-activity genotypes of GSTM1, GSTT1 and GSTP1 were associated with a 19-48% increase in odds ratio (OR) of bladder cancer. The strongest association was noted for GSTM1 (OR for the null genotype=1.48, 95% confidence interval CI=1.19-1.83). When the three GST genes were examined together, there was a monotonic, statistically significant association between increasing number of null/low-activity genotypes and risk (P for trend=0.002). OR (95% CI) for one and 2 or more null/low-activity GST genotypes was 1.42 (1.12-1.81) and 1.71 (1.25-2.34), respectively, relative to the absence of null/low-activity GST genotype. NAT2 slow acetylation was associated with increased bladder cancer risk (OR=1.26, 95% CI=1.00-1.57, P=0.048). The joint effect of NAT2 slow acetylator and null/low-activity GST genotypes on bladder cancer risk became stronger (OR=2.27, 95% CI=1.32-3.91), especially among individuals with known high exposures to carcinogenic arylamines. Among subjects with known low exposures to carcinogenic arylamines, the NAT2 slow acetylation-bladder cancer association was apparent only in the presence of 2 or more null/low-activity GST genotypes. There were no associations between bladder cancer risk and NAT1 genotype or CYP1A2 phenotype. 2) In utero PAH Exposure Alters CpG Methylation Status in Neonates (collaboration with R. Perera, Columbia U.;Pittman et al, Poster). We have previously reported that maternal exposure to ambient polycyclic aromatic hydrocarbons (PAHs) was associated with PAH-DNA adducts in neonatal cord blood and post-natal developmental endpoints. High in utero PAH exposure produced higher PAH-DNA adducts in cord blood than maternal blood suggesting a higher biologically effective dose in the neonates. Although in vitro studies suggest PAH exposure can affect CpG methylation, few human in vivo studies related to methylation have been carried out. Therefore, we have conducted a hypothesis-generating pilot study examining CpG methylation in white blood cell DNA from 12 mother-child pairs from Krakow, Poland, evenly divided between mothers with high and low ambient PAH exposure and an almost even balance of male and female neonates. We measured methylation in triplicate using Illuminas Methylation Cancer Panel I containing 1505 CpG probes, representing 808 genes. We removed from analyses methylation probes near SNPs and those that had poor performance with reference standards. We compared CpG methylation across exposure groups, applying t-tests adjusted for false discovery rate and identified significant differences in methylation associated with maternal PAH exposure. We also selected a subset of 10 CpG sites in 9 biologically-significant genes to test using Pyrosequencing, an alternate technology for measuring methylation. PAH exposure effects were detected among all subjects and within each subgroup (mothers, male and female neonates), but each group showed distinct PAH-related methylation differences. For most CpGs, PAH-associated differences were not consistent between mothers and neonates, suggesting PAH effects may be different in mothers and neonates. Mothers with high PAH exposure had 160 CpG sites in 152 genes (59% hypomethylated) that differed significantly relative to mothers with low exposure. High exposure female neonates had 137 altered CpG sites with 55% hypomethylated;male neonates, 85 sites, 51% hypomethylated. Among high-PAH exposure neonates (both males and females), 27 CpG sites had significantly different methylation levels relative to neonates with low exposure. Interestingly, among neonates, many sites that were hypermethylated in females were hypomethylated in males, and vice versa. Comparing pyrosequencing with Illumina Goldengate results we observed on average a 15% difference between platforms, with the Illumina results showing a higher level of methylation. We performed a cross platform correlation analysis for the 10 assays and found a large range of correlations, some poor. We hypothesize the difference reflects the different technologies employed in the two assays, consistent with a recently published study comparing the two technologies (54). Among all subjects and across all strata, hypomethylation was most commonly associated with high in utero PAH exposure.